When penicillin first became widely available it was a medical miracle. With penicillin, doctors were able to treat disease that was previously untreatable and often lethal. Soon, drug companies were mass-producing penicillin and doctors were giving it to patients frequently.
However, shortly after the start of widespread use of penicillin, bacteria began to appear that were resistant to the antibiotic. The first microbe to become resistant to penicillin was Staphylococcus aureaus. This bacterium is generally part of the harmless resident flora of the human body. However, it can also cause illness such as pneumonia.
Other bacteria began to show resistance to penicillin, and new antibiotics were discovered and introduced. With almost every introduction of a new antibiotic, bacteria have evolved a mechanism to resist the drug. Thus, bacteria have been found that are resistant to methicillin, oxacillin, chloramphenicol, neomycin, terramycin, tetracycline, and cephalosporins. Many bacteria are resistant to a number of types of antibiotics. Moreover, resistant bacteria can transfer the resistance gene to other bacteria—even bacteria of dissimilar species.
Despite the development of bacterial resistance to antibiotics, researchers have been able to discover new agents to battle the bugs. However, many of these new agents can also be toxic to humans. Moreover, the new agents may kill most of the resident microflora of the gastrointestinal tract, making the patient more susceptible to the invasion of disease causing bacteria.
Vancomycin has been used as the drug of last resort in treating multiple-resistant bacterial strains. However, bacterial strains have been discovered that resist vancomycin, and many medical experts fear that we will be without weapons to fight resistant bacteria.
Similar to the problems facing the treatment of bacterial disease are those associated with herbicidal agents. Herbicidal agents have been used by farmers, gardeners, and others for many years to combat undesirable plants and to increase crop yields. However, as with antibiotics, some plants have developed resistance to these compounds. Additionally, recent environmental regulations have caused some of the most effective herbicides to be discontinued because of potential harm to the environment and humans. Thus, there is a need to provide new herbicidal compounds that are less damaging to the environment and yet effective at killing undesirable plants.
One potential target for finding compounds that are effective antibacterial and/or herbicidal agents is the pathways for the synthesis of isoprenoid compounds. Isoprenoid compounds form a large ubiquitous class of natural products consisting of over 30,000 individual members. They fulfill a wide variety of cellular functions—such as components of cell membranes (sterols), electron transport (ubiquinones), signal transduction (prenylated proteins), photosynthetic pigments (chlorophylls), and cell wall biosynthesis (dolichols)—essential for viability.
Until recently, all isoprenoid compounds were thought to be synthesized from acetyl-CoA by the widely accepted mevalonate (MVA) pathway found in eukaryotes and archaebacteria. Work stimulated by labeling patterns in bacterial hopanoids and ubiquinones inconsistent with their biosynthesis by the MVA pathway led to the discovery of a new independent route to these molecules in many bacteria, green algae, and plants. In the newly discovered pathway, the five carbon atoms in the basic isoprenoid unit are derived from pyruvate and D-glyceraldehyde 3-phosphate (GAP). Pyruvate and GAP are condensed to give 1-deoxy-D-xylulose 5-phosphate (DXP).
In addition to serving as a precursor for the biosynthesis of thiamine and pyridoxol, DXP undergoes rearrangement and reduction to form 2-methylerythritol 4-phosphate (MEP), the first committed intermediate in the MEP pathway for biosynthesis of isoprenoids. MEP is then condensed with CMP to form a cytidine derivative, followed by phosphorylation of the C2 hydroxyl group and elimination of CMP to form a 2,4-cyclic diphosphate. The remaining steps to the fundamental five-carbon isoprenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), have not been established. For most organisms reported to date, the enzymes in the MEP pathway are encoded by essential single copy genes. The exception is a Streptomyces gram-positive strain, which contains both the MEP and MVA pathways.
DXP synthase (DXPase) lies just before the branch point to the B vitamins and isoprenoids. Genes encoding the enzyme have been cloned from a number of species, including E. coli, Streptomyces sp. strain CL190, Mentha x piperiia, and Capsicum annuum. Disruption of the E. coli dxs gene is lethal. DXPase catalyzes the decarboxylation of pyruvate and the subsequent condensation of the thiamine bound two-carbon intermediate with GAP in a reaction similar to those catalyzed by transketolases. Interestingly, DXPases contain regions with strong homology to the E1 subunit of pyruvate dehydrogenases and to transketolases. Although recombinant forms of the enzyme can use either GAP or D-glyceraldehyde as co-substrates with pyruvate, the phosphorylated form of the deoxy-sugar appears to be the normal intermediate in the pathway.
In light of the foregoing it would be a significant advancement in the art to provide a new method for screening for antibacterial and herbicidal agents. It would be a further advancement if the method produced compounds that were not toxic to humans and had decreased effect on the environment. An additional advancement would be to provide compounds that were effective antibiotics and herbicides. It would also be a significant advancement if an organism could be engineered for use in screening agents for activity against a large number of plants and bacteria. It would be a further advancement if the method could exploit the MEP pathway which is present in plants and eubacteria. Such organisms, compounds, and methods are disclosed and claimed herein.